The advent of DNA microarray technology makes it possible to build an array of hundreds of thousands of DNA sequences in a very small area, such as the size of a microscopic slide. See, e.g., U.S. Pat. Nos. 6,375,903 and 5,143,854, each of which is hereby incorporated by reference in its entirety. The disclosure of U.S. Pat. No. 6,375,903 enables the construction of so-called maskless array synthesizer (MAS) instruments in which light is used to direct synthesis of the DNA sequences, the light direction being performed using a digital micromirror device (DMD). Using an MAS instrument, the selection of DNA sequences to be constructed in the microarray is under software control so that individually customized arrays can be built to order. In general, MAS based DNA microarray synthesis technology allows for the parallel synthesis of over 800,000 unique oligonucleotides in a very small area of on a standard microscope slide. For many applications, the entirety of the synthesized array is devoted to the evaluation of one sample of test nucleotides. In these applications, the entire microarray area is enclosed in a small chamber so as to allow for the application of the single sample, thus providing a very efficient means for measuring the expression level of a very large number of genes within that one sample. A typical application of this sort is gene expression profiling.
The availability of microarrays is revolutionizing the way that researchers collect data about the expression of genes in cells and organisms. By proper selection of the sequence of probes, the profile of gene expression in a cell or tissue can be revealed. Since microarrays can have hundreds of thousands of features, each feature having a set of identical DNA probes, the microarray can by used to collect a massive amount of data in parallel. For example, DNA microarray technology has been applied to many areas such as gene expression and discovery, mutation detection, allelic and evolutionary sequence comparison, and genome mapping. For some applications, the amount of data gathering potential in a microarray is simply too much, since sometimes the data to be collected involves far fewer probes than a microarray's full capacity.
In applications, it is desired to study a smaller number of genes. In some of such applications it is desired to test a large number of samples against a smaller set of probes that the full capacity of the microarray makes available. To perform studies for these applications, the microarray can be logically divided into any number of smaller sub-arrays each having the same or similar nucleotide probes, a concept sometimes referred to as an array of arrays. Instead of a single microarray, for example, with 100,000 features or probe areas, the microarray can be divided into 1000 sub-arrays, each of 100 features. To use an array of arrays efficiently, multiple samples are hybridized in parallel, in a single experiment, with each sample being hybridized to a given and predetermined area of the microarray, an area making up one of the sub-arrays in the array of arrays. This parallel loading strategy provides for efficient utilization of the high synthesis capacity of the microarray. In order to load multiple samples onto a microarray made up of sub-arrays, while avoiding sample cross-contamination, some mechanism must be provided to prevent leakage of each sample to adjacent sub-arrays. Currently, microarrays built for this purpose (e.g., U.S. Pat. No. 5,874,219) use physical wells to separate probe sets for different samples. This approach may not be optimal for array of array microarrays having a large number of sub-arrays, where alignment of physical wells with the sub-arrays could be challenging.
Another of the technical challenges arising from the use of sub-arrays is the delivery and control of sample volumes delivered to each of the small sub-arrays. It is envisioned that sample volumes may fall as low as 200 nl per sample with sub-arrays. When sample sizes are this small, evaporation of liquids and delivery of samples both become serious problems. This invention describes a method and apparatus to overcome the problems of sample delivery to the sub-arrays, and provides for a methodology to conduct hybridization reactions in small volumes on the arrays of arrays.